Petroleum is the primary feedstock, not only for the fuels we use, but the majority of the chemicals we consume as well. The chemical industry is heavily reliant on this non-renewable resource. Replacement of petroleum with renewable feedstocks ensures longer-term viability and environmental sustainability. Novel fermentation based processes to make chemicals have been a contributing technology, enabling the change to renewable feedstocks (Werpy & Peterson, Top Value Added Chemicals from Biomass. Volume I—Results of Screening for Potential Candidates from Sugars and Synthesis Gas., Yixiang et al. “Green” Chemicals from Renewable Agricultural Biomass—A Mini Review. The Open Agriculture Journal, 2008). These fermentation processes have made rapid advancements in recent years due to technology developments in the fields of fermentation science, synthetic biology, as well as metabolic and enzyme engineering (Jarboe, L. R., et al., Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology. J Biomed Biotechnol, 2010, Lee, J. W., et al., Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat Chem Biol, 2012). Despite these substantial advances, most successful examples of rationale directed engineering approaches have also greatly relied on numerous cycles of trial and error. The field of metabolic engineering has historically been limited in predicting the behavior of complex biological systems in-vivo, from simplified models and basic in-vitro biochemical principles. Such rational approaches have required significant a priori knowledge of microbial physiology that in many cases is incomplete. This is particularly true for complex phenotypes that require an intricate balance between the activities of many seemingly unrelated gene products. In many cases it has proven much more difficult than expected to integrate a possibly well characterized production pathway into a living host and balance the complex requirements of both biomass growth and production.
One solution is the development of platform microbial strains that utilize synthetic metabolic valves (SMVs) that can decouple growth from product formation. These strains enable the dynamic control of metabolic pathways, including those that when altered have negative effects on microorganism growth. Dynamic control over metabolism is accomplished via a combination of methodologies including but not limited to transcriptional silencing and controlled enzyme proteolysis. These microbial strains are utilized in a multi-stage bioprocess encompassing as least two stages, the first stage in which microorganisms are grown and metabolism can be optimized for microbial growth and at least one other stage in which growth can be slowed or stopped, and dynamic changes can be made to metabolism to improve production of desired product, such as a chemical or fuel. The transition of growing cultures between stages and the manipulation of metabolic fluxes can be controlled by artificial chemical inducers or preferably by controlling the level of key limiting nutrients. In addition, genetic modifications may be made to provide metabolic pathways for the biosynthesis of one or more chemical or fuel products. Also, genetic modifications may be made to enable the utilization of a variety of carbon feedstocks including but not limited sugars such as glucose, sucrose, xylose, arabinose, mannose, and lactose, oils, carbon dioxide, carbon monoxide, methane, methanol and formaldehyde.
This approach allows for simpler models of metabolic fluxes and physiological demands during a production phase, turning a growing cell into a stationary phase biocatalyst. These synthetic metabolic valves can be used to turn off essential genes and redirect carbon, electrons and energy flux to product formation in a multi-stage fermentation process. One or more of the following enables these synthetic valves: 1) transcriptional gene silencing or repression technologies in combination with 2) inducible enzyme degradation and 3) nutrient limitation to induce a stationary or non-dividing cellular state. SMVs are generalizable to any pathway and microbial host. These synthetic metabolic valves allow for novel rapid metabolic engineering strategies useful for the production of renewable chemicals and fuels and any product that can be produced via whole cell catalysis.
A simplified two-stage bioprocess using synthetic metabolic valves is depicted in FIG. 1, strains are grown in a minimal media with a single limiting nutrient such as inorganic phosphate. During this growth phase cells are not producing any product other than biomass and as a result are not subject to any possible toxic or unwanted side effects of product formation. Biomass growth and yield can be optimized. As the limiting nutrient is depleted, cell growth is stopped. Simultaneously, these strains will be engineered to contain synthetic metabolic valves, which silence genes and enzymes essential for growth and redirect carbon, electrons and energy to any molecule of interest. This process utilizes a novel combination of a two-stage production and concurrent metabolic engineering strategy.
There is significant precedent in the biotechnology industry for using and scaling two stage processes similar to that described in FIG. 1. Many similar processes are routinely used for the heterologous expression of proteins. In these standard processes cells are grown to a productive or “primed” state for protein synthesis (such as mid-exponential phase in E. coli) and then induced to express a heterologous protein. In many cases, the diversion of cellular amino acids and energy to the heterologous protein has a significant effect on, if not halting, cellular growth. It is not surprising that these types of processes have not been developed for the biological production of small molecules as historically most successful efforts to metabolically engineer the production of small molecules have leveraged the power of anaerobic metabolism to couple product formation with growth.
Anaerobic growth-coupled product formation enables the use of powerful growth based selections to identify better producers. The faster the cells grow the more product they make. This has allowed for the classical selection of industrial strains for many natural products such as ethanol and isobutanol. However, the requirement for anaerobic production greatly limits the number and variety of different molecules or products that can be made using synthetic biology. Numerous products would require aerobic metabolism to supply the needed energy and cofactors to allow for a thermodynamically feasible metabolic pathway. In these cases a generic and robust aerobic production platform would greatly simplify the optimization and scale up of a diverse number of products. A controlled multi-stage process, enabled by synthetic metabolic valves, supplies such a platform.
Synthetic metabolic valves enable synthetic biologists and metabolic engineers the ability to decouple the complex metabolic and thermodynamic needs of growth from those of product formation. This decoupling also enables the removal of growth based regulatory mechanisms that may inhibit product formation and allows for the silencing of essential metabolic pathways that may detract from or interfere with production. These essential interfering metabolic pathways could include amino acid biosynthesis or the citric acid cycle as well as the biosynthesis of many secondary metabolites, and those pathways involved in maintaining intracellular redox and energy balances. These pathways have traditionally been off limits to many metabolic engineering strategies, as attempts at manipulation have led to growth defects.